Heat Inactivation Method Research Articles

Application of Bacillus Coagulans as Paraprobiotic Against Acute Hepatopancreatic Necrosis Disease in Shrimp.

Paraprobiotics, known as non-viable or ghost probiotics, have attracted attention for their benefits over live microbial cells. This study was designed to investigate the paraprobiotic effects of heat-killed Bacillus coagulans on the white leg shrimp Litopenaeus vannamei. The paraprobiotic formulation was prepared in three different concentrations including B. coagulans 1 (107 cells g-1 diet), B. coagulans 2 (108 cells g-1 diet), and B. coagulans 3 (109 cells g-1 diet) through heat inactivation method. Preliminary toxicity assessments revealed that post-larvae shrimps (mean weight ± SE: 0.025 ± 0.007g) treated with B. coagulans 1, 2 and 3 paraprobiotic formulations exhibited no mortality, confirming the non-toxic nature of the formulated diet. In a 90-day feeding trial involving juvenile shrimps (mean weight ± SE: 0.64 ± 0.05g), growth parameters and feed conversion ratios improved in all experimental groups. Subsequently, these shrimps were challenged with Vibrio parahaemolyticus, revealing that paraprobiotic-fed shrimps exhibited significant survival rate improvements. Oxidative stress-related enzyme activities, such as superoxide dismutase and catalase, increased in paraprobiotic-fed shrimps post-Vibrio challenge, while the challenged control group showed decreased activity (p < 0.001). Nitric oxide levels are also increased in paraprobiotic-treated shrimp, with B. coagulans 3 showing a significant rise in nitric oxide activity (p < 0.001). This study further demonstrated the positive impact of paraprobiotic treatment on digestive enzymes, immune-related parameters (e.g., total hemocyte count, prophenoloxidase, and respiratory burst activity), and overall disease resistance. These findings suggest that B. coagulans paraprobiotics have the potential to enhance antioxidant, antibacterial, and immune-related responses in L. vannamei, making them a valuable addition to shrimp aquaculture.

PLACENTALALKALINE PHOSPHATASE AS A THRESHOLD CONCEPT FOR EARLY DETECTION OF PRIMARY LUNG CANCER

The biochemical existing tool of diagnostic methods to lung cancer cases need to be improved. In order to validate an early screening of primary tumor patients, a developed a simple procedure or technique was demanded. The aims of this study were to provide an overview of alkaline Placental Alkaline Phosphatase activity in lung cancer. Using heating inactivation method regarding the measurement of Placental Alkaline Phosphatase activity as an early diagnosis marker in lung cancer cases. Total alkaline phosphatase and Placental alkaline phosphatase activity were measured in patients of Lung cancer patients who were classified according to the site of tumor by histological picture. ALP isoenzymes were identified by heat inactivation, and compared with the most frequently applied method (ELISA). Monitoring of the Total ALPand Placental ALPactivity in the studied groups using two different methods were shown a highly performance of heating method by an experimental assessment to confirm the accuracy and validity of the proposed method. The distribution of serum placental ALP isoenzyme activity in patients and control groups which was measured by two different methods were found to be (20.2-43.1) IU/Lrespectively (measured by heating method) and (394.3- 454.5) pg/mLmeasured by ELISA method) respectively. Placental ALP isoenzyme showed a high significant activity in lung cancer patients than healthy control with p value less than (0.05). That application of the heat inactivation method yields similar indication to the ones obtained by the highly and specific enzyme-linked immunosorbent assay. The results of detection Placental alkaline phosphatase in serum were in excellent agreement and could have a potentially extensive application for Placental alkaline phosphatase quantification.

Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis.

ABSTRACTStudies involving the pathogenic organism Mycobacterium tuberculosis routinely require advanced biosafety laboratory facilities, which might not be readily available in rural areas where tuberculosis burdens are high. Attempts to adapt heat inactivation techniques have led to inconsistent conclusions, and the risk of protein denaturation due to extensive heating is impractical for subsequent mass spectrometry (MS)-based protein analyses. In this study, 240 specimens with one or two loops of M. tuberculosis strain H37Rv biomass and specific inactivated solutions were proportionally assigned to six heat inactivation methods in a thermal block at 80°C and 95°C for 20, 30, and 90 min. Twenty untreated specimens served as a positive control, and bacterial growth was followed up for 12 weeks. Our results showed that 90 min of heat inactivation was necessary for samples with two loops of biomass. Further protein extraction and a matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS assay demonstrated adequate scores for bacterial identification (≥1.7), with the highest score achieved in the 80°C/90 min and 95°C/30 min treatment groups. A proteomics study also confidently identified 648 proteins with ∼93% to 96% consistent protein abundances following heating at 95°C for 20, 30, and 90 min. Heat inactivation at 95°C for 90 min yielded the most quantifiable proteins, and a functional analysis revealed proteins located in the ribosomal subunit. In summary, we proposed a heat inactivation method for the M. tuberculosis strain H37Rv and studied the preservation of protein components for subsequent bacterial identification and protein-related assays.IMPORTANCE Inactivation of Mycobacterium tuberculosis is an important step to guarantee biosafety for subsequent M. tuberculosis identification and related research, notably in areas of endemicity with minimal resources. However, certain biomolecules might be denatured or hydrolyzed because of the harsh inactivation process, and a standardized protocol is yet to be determined. We evaluated distinct heating conditions to report the inactivation efficiency and performed downstream mass spectrometry-based M. tuberculosis identification and proteomics study. The results are important and useful for both basic and clinical M. tuberculosis studies.

A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection.

A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13 Assisted Saliva-based & Smartphone Integrated Testing), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.

Proposition of a safe Mycobacterium tuberculosis complex denaturation method that does not compromise the integrity of DNA for whole-genome sequencing

Whole-genome sequencing plays now a leading role in epidemiologic studies of tuberculosis. DNA extraction of Mycobacterium tuberculosis complex (MTBC) requires complete inactivation of the strains, to be handled for further molecular procedures. In this study we compared two chloroform-based denaturation methods (one with a step of heat killing, one without) to a traditional heat inactivation method. Our results showed that 40% of the strains of MTBC treated by the traditional protocol resulted in a positive culture whereas no culture was observed with the two chloroform-based protocols. The DNA extracts obtained with chloroform-based protocols preparation were successfully used for whole-genome sequencing. We recommend inactivation with our rapid and efficient denaturation method using chloroform without heat killing which met our expectations and biosecurity requirements.

New short-term heat inactivation method of cytomegalovirus (CMV) in breast milk: impact on CMV inactivation, CMV antibodies and enzyme activities

ObjectivesBreast milk (BM) is the primary source of cytomegalovirus (CMV) transmission to premature infants with potentially harmful consequences. We therefore wanted to evaluate temperature and duration of short-term BM pasteurisation...

Expanding the spectrum of HEXA mutations in Indian patients with Tay–Sachs disease

Tay–Sachs disease is an autosomal recessive neurodegenerative disorder occurring due to impaired activity of β-hexosaminidase-A (EC 3.2.1.52), resulting from the mutation in HEXA gene. Very little is known about the molecular pathology of TSD in Indian children except for a few mutations identified by us. The present study is aimed to determine additional mutations leading to Tay–Sachs disease in nine patients confirmed by the deficiency of β-hexosaminidase-A (<2% of total hexosaminidase activity for infantile patients) in leucocytes. The enzyme activity was assessed by using substrates 4-methylumbelliferyl-N-acetyl-β-d-glucosamine and 4-methylumbelliferyl-N-acetyl-β-d-glucosamine-6-sulfate for total-hexosaminidase and hexosaminidase-A respectively, and heat inactivation method for carrier detection. The exons and exon–intron boundaries of the HEXA gene were bi-directionally sequenced on an automated sequencer. ‘In silico’ analyses for novel mutations were carried out using SIFT, Polyphen2 and MutationT@ster software programs. The structural study was carried out by UCSF Chimera software using the crystallographic structure of β-hexosaminidase-A (PDB-ID: 2GJX) as the template. Our study identified four novel mutations in three cases. These include a compound heterozygous missense mutation c.524A>C (D175A) and c.805G>C (p.G269R) in one case; and one small 1bp deletion c.426delT (p.F142LfsX57) and one splice site mutation c.459+4A>C in the other two cases respectively. None of these mutations were detected in 100 chromosomes from healthy individuals of the same ethnic group. Three previously reported missense mutations, (i) c.532C>T (p.R178C), (ii) c.964G>T (p.D322Y), and (iii) c.1385A>T (p.E462V); two nonsense mutations (i) c.709C>T (p.Q237X) and (ii) c.1528C>T (p.R510X), one 4bp insertion c.1277_1278insTATC (p.Y427IfsX5) and one splice site mutation c.459+5G>A were also identified in six cases. We observe from this study that novel mutations are more frequently observed in Indian patients with Tay–Sachs disease with clustering of ~73% of disease causing mutations in exons 5 to 12. This database can be used for a carrier rate screening in the larger population of the country.

Values of alkaline phosphathase and their isoenzyme profiles in patients with cancer in respect to bone and liver metastasis

Background: Alkaline phosphatase is a glycoprotein that catalyzes two kinds of chemical reactions: hydrolysis of phosphorus ester breaking P-O bonds and phospho-transfer reactions in which phosphoric group is transferred to an acceptor molecule. In the human body, ALP exists in multiple molecular forms whose heterogeneity is partly due to genetic factors and partly to posttranslational modifications. The aim was to evaluate a total ALP activity and its isoforms in cancer patients with bone and liver metastasis in comparison to healthy controls. Methods: Human serum was collected from 20 healthy individuals, and 20 cancer patients with bone and liver metastases, with metastases confirmed by ultrasound, computerized tomography and a radiology scan. Determination of ALP was done by the endpoint spectrophotometric method. Isoenzymes were determined by heat inactivation method. Results: In cancer patients, the total ALP activity was significantly higher (p&lt; 0.05) compared to healthy controls. In the sera of cancer patients with liver metastases, the remaining ALP activity was two-fold higher in comparison to bone metastases. Conclusion: Determination of ALP isoenzymes is important but a correct clinical interpretation in the context of other analyses is vital for a proper diagnosis of a disease.

Identification of novel mutations in HEXA gene in children affected with Tay Sachs disease from India.

Tay Sachs disease (TSD) is a neurodegenerative disorder due to β-hexosaminidase A deficiency caused by mutations in the HEXA gene. The mutations leading to Tay Sachs disease in India are yet unknown. We aimed to determine mutations leading to TSD in India by complete sequencing of the HEXA gene. The clinical inclusion criteria included neuroregression, seizures, exaggerated startle reflex, macrocephaly, cherry red spot on fundus examination and spasticity. Neuroimaging criteria included thalamic hyperdensities on CT scan/T1W images of MRI of the brain. Biochemical criteria included deficiency of hexosaminidase A (less than 2% of total hexosaminidase activity for infantile patients). Total leukocyte hexosaminidase activity was assayed by 4-methylumbelliferyl-N-acetyl-β-D-glucosamine lysis and hexosaminidase A activity was assayed by heat inactivation method and 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulphate lysis method. The exons and exon-intron boundaries of the HEXA gene were bidirectionally sequenced using an automated sequencer. Mutations were confirmed in parents and looked up in public databases. In silico analysis for mutations was carried out using SIFT, Polyphen2, MutationT@ster and Accelrys Discovery Studio softwares. Fifteen families were included in the study. We identified six novel missense mutations, c.340 G>A (p.E114K), c.964 G>A (p.D322N), c.964 G>T (p.D322Y), c.1178C>G (p.R393P) and c.1385A>T (p.E462V), c.1432 G>A (p.G478R) and two previously reported mutations. c.1277_1278insTATC and c.508C>T (p.R170W). The mutation p.E462V was found in six unrelated families from Gujarat indicating a founder effect. A previously known splice site mutation c.805+1 G>C and another intronic mutation c.672+30 T>G of unknown significance were also identified. Mutations could not be identified in one family. We conclude that TSD patients from Gujarat should be screened for the common mutation p.E462V.

Voltammetric behavior of the heat-treating PC-3 cells and its application in drug sensitivity test

A novel and simple strategy for improving the voltammetric response of the cells by heat inactivation method had been proposed. By comparing the voltammetric behavior of the intact cell suspension with that of the fragmentized cell suspension, the results indicated that the former was weaker than the latter, and heat inactivation was superior to ultrasonication in improving the voltammetric response of the cells by increasing the release of the intracellulare guanine and xanthine into the cell eluent as a result of fragmenting the cell membrane. The comparison of cytotoxicity evaluations of the PC-3 cells based on the electrochemical method and the MTT assays suggested the antitumor drug sensitivity test based on the electrochemical method was credible, sensitive, rapid, inexpensive and objective.

Immunogenicity of Factor VIII: Is Clotting the “Danger” Signal?.

It is well established that mice lacking factor VIII (E16 or fVIII−/−) respond to intravenous (i.v.) or intraperitoneal (i.p.) injections of therapeutic doses of human fVIII in PBS. This leads to both T-cell priming for cytokine production and inhibitor formation against fVIII with specificity similar to that of hemophilia A patients (Pratt et al., 2004). The lack of tolerance to murine fVIII may be important in the ability of fVIII−/− mice to respond to human fVIII. Nonetheless, the ability to respond effectively in the absence of extrinsic adjuvant begs the question of what is the “danger signal” required for immune responsiveness to fVIII. Even more surprising is the fact that mice with normal hemostasis respond to therapeutic doses of human fVIII given intravenously (i.v.), a normally tolerogenic route (Lin, Soukhareva, and Scott, ASH 2004). We proposed to investigate whether fVIII has any intrinsic adjuvanticity. Preliminary experiments failed to support the notion that fVIII was a polyclonal B cell activator or could stimulate up regulation of MHC class II or co-stimulatory molecules in vitro or in vivo. We hypothesized that the immunogenicity of fVIII is due to its ability to initiate clotting and that this cascade leads to local co-stimulatory events. To test this, fVIII−/− (C57Bl/6 background) or normal Balb/c mice were injected with either native (N) fVIII or heat inactivated (HI) fVIII (56o, 30′). This heat inactivation method was verified to destroy biologic activity of fVIII in chromogenic assays. After five injections, the antibody response elicited by NfVIII was three- to fourfold higher than that elicited by HIfVIII. ELISA assays with monoclonal antibodies against different fVIII domains (a gift of Dr. Pete Lollar) indicated a partial loss of binding to conformational B cell epitopes in HIfVIII. T cell proliferation to both preparations of fVIII demonstrated conservation of T cell epitopes. Importantly, the T cell response to fVIII was dramatically reduced in the group immunized with HIfVIII. These results suggest that the ability of fVIII to initiate a localized clotting event plays a critical role in the immunogenicity of fVIII.(Supported by NIH grants R01 HL061883 and T32 HL07698).

16. Late-onset GM2 gangliosidosis and other hexosaminidase mutations among Jews

Publisher Summary This chapter discusses the late-onset G M2 gangliosidosis and other Hexosaminidase mutation among Jews. Tay-Sachs Disease (TSD) is caused by absence of β-hexosaminidase (Hex) A, following which heat inactivation method (HIM) for quantifying Hex A using the synthetic substrate 4-MUG. The interaction between Hex A and G M2 ganglioside substrate is accomplished by a nonenzymatic protein, the G M2 activator. Hex A-deficient adults have residual Hex A activity that are significantly higher in TSD patients, but significantly lower than in TSD heterozygotes. AGG is a slowly progressing neurodegenerative disorder. The patients have very low Hex A activity and usually carry the Gly269Ser mutation in compound heterozygosity with an infantile TSD mutation. Homozygotes for the Gly269Ser mutation are only mildly affected. The enzyme β-hexosaminidase (Hex) B does not degrade the G M2 ganglioside in humans, and therefore has no direct involvement in TSD. However, it is a major participant in the routine enzymatic assay for identification of TSD genotypes because Hex B, like Hex A, hydrolyzes the synthetic substrate 4-MUG, and, unlike Hex A, it is heat stabile. It is estimated that 1 in 140 Moroccans is a TSD carrier, and seven different TSD mutations in this community have been identified.

Immunological properties of recombinant hepatitis B surface antigen expressed in mammalian cell (C127).

We examined the immunological properties of the recombinant hepatitis B surface antigen (r-HBsAg) which was expressed in mammalian cell (C127). The cross-immunity of r-HBsAg and plasma-derived hepatitis B surface antigen (p-HBsAg) were tested using Western blotting and ELISA with guinea pig polyclonal antibody and naturally infected human-derived antibody and the both antigens show the same results in their response pattern and intensity, which indicate they have a good cross-immunity. From the measurement of ED50 after formalin- or heat-inactivation, both r-HBsAg and p-HBsAg showed ED50 of 0.2-0.3 in formalin-inactivation, while r-HBsAg was 0.05-0.09 and p-HBsAg was 0.03-0.07 in heat-inactivation, which means heat-inactivation method is 3-4 times superior in immunogenicity. In the immunopersistency test performed in guinea pig for the period of 3 months with two different adjuvants, antibody titer was 34.2 with muramyl dipeptide adjuvant, which was 1.8 times greater than the antibody titer of 18.9 with AlPO4 adjuvant. The mutagenicity of r-HBsAg, examined by Ames test and micronucleus test, were all negative. In conclusion, r-HBsAg has the same cross-immunity with p-HBsAg, and heat-inactivation method and muramyl dipeptide adjuvant allow development of r-HBsAg vaccine with excellent immunogenicity.

Unusual thermolability properties of beta-hexosaminidase: studies of enzyme from cultured cells and clinical implications.

Tay-Sachs disease (TSD) is a neurodegenerative genetic disorder caused by a deficiency of beta-hexosaminidase A (Hex A) activity. To diagnose TSD and to screen for TSD heterozygosity, laboratories use an assay that exploits the differential thermolability of the major beta-hexosaminidase isoenzymes, Hex A and Hex B. At 50-52 degrees C Hex A is labile, and Hex B is stable. We previously noted that the stability of leukocyte Hex B at 52 degrees C varied significantly, depending on the sample concentration in the incubation mixture. We have now examined this phenomenon in enzyme from cultured cells used for prenatal and postnatal diagnostic testing. We found that fibroblast Hex A and Hex B behave similarly to the leukocyte isoenzymes. In control and TSD fibroblasts there was a linear correlation between Hex B thermostability and sample concentration; at lower sample concentrations Hex B was less stable than at higher concentrations. Dialysis of the samples prior to heat treatment did not change the thermostability properties of Hex B, indicating that the change in stability is not due to a soluble low molecular weight substance. Cultured amniotic fluid cell and chorionic villus cell Hex B had a similar, but less pronounced, instability at low sample concentrations. Therefore, the unusual thermolability properties of Hex B, first detected for leukocyte Hex B, were noted in multiple tissues. Based on these data, we suggest that the concentration of cell extract be stringently controlled when the heat-inactivation method is used for the pre- or postnatal diagnosis of TSD, and that supplementation with non-thermolability-based beta-hexosaminidase assays should be employed as needed.

P.3.052 - First episode schizophrenia: Treatment and outcome

This chapter discusses the late-onset GM2 gangliosidosis and other Hexosaminidase mutation among Jews. Tay-Sachs Disease (TSD) is caused by absence of β-hexosaminidase (Hex) A, following which heat inactivation method (HIM) for quantifying Hex A using the synthetic substrate 4-MUG. The interaction between Hex A and GM2 ganglioside substrate is accomplished by a nonenzymatic protein, the GM2 activator. Hex A-deficient adults have residual Hex A activity that are significantly higher in TSD patients, but significantly lower than in TSD heterozygotes. AGG is a slowly progressing neurodegenerative disorder. The patients have very low Hex A activity and usually carry the Gly269Ser mutation in compound heterozygosity with an infantile TSD mutation. Homozygotes for the Gly269Ser mutation are only mildly affected. The enzyme β-hexosaminidase (Hex) B does not degrade the GM2 ganglioside in humans, and therefore has no direct involvement in TSD. However, it is a major participant in the routine enzymatic assay for identification of TSD genotypes because Hex B, like Hex A, hydrolyzes the synthetic substrate 4-MUG, and, unlike Hex A, it is heat stabile. It is estimated that 1 in 140 Moroccans is a TSD carrier, and seven different TSD mutations in this community have been identified.

Improved immunoturbidimetric method for rheumatoid factor testing.

The performance of two immunoturbidimetric modifications for rheumatoid factor (RF) testing, which differ with respect to the means of complement inactivation (heat treatment and inactivation with polyvinyl sulphonate), were compared in serum samples from 87 patients with rheumatoid arthritis (RA) and from 403 healthy subjects. IgM-rheumatoid factor titres were also measured with an enzyme linked immunosorbent assay (ELISA). Both immunoturbidimetric tests gave positive reactions (rheumatoid factor > or = 20 IU/ml) in 74 out of the 87 (85%) RA sera. In cases with high RF concentrations the results after chemical inactivation tended to be slightly higher compared with heat inactivation. In healthy subjects rheumatoid factor was detected in 19/403 (4.7%) sera using heat inactivation and in 22/403 (5.5%) sera with chemical inactivation of complement. Interrun coefficient of variation in the chemical inactivation assay was 4.4%; with the heat inactivation method it was 8.1%. In the ELISA, a marginally better correlation was noted in the results obtained using chemical inactivation. Inactivation of complement by means of polyvinyl sulphonate offers the advantage of easier test performance and better reproducibility, and the results may reflect more accurately true rheumatoid factor concentrations.

Thermolabile Hexosaminidase (Hex) B: Diverse Frequencies among Jewish Communities and Implication for Screening of Sera for Hex A Deficiencies

Twenty unrelated people with thermolabile beta-hexosaminidase (Hex) B were identified in random samples of 41,561 adult Israeli Jews whose sera were screened for Hex A levels. Eighteen of them originated from contiguous Middle Eastern countries (n = 1,337) and only 2 were Ashkenazi Jews (n = 38,388). None was found among the screened Moroccan Jews (n = 1,524). The commonly used screening test for detection of Tay-Sachs disease (TSD) carriers is the serum Hex heat inactivation method (HIM), which is based on the assumption that Hex A is the only thermolabile component of Hex; However, in the presence of thermolabile Hex B, HIM could lead to false-negative results. Since TSD is very rare among Mideastern Jews while thermolabile Hex B is very rare among Ashkenazi and Moroccan Jews, it is concluded that at present there is almost no risk of such false-negative results. In order to further reduce this risk it is recommended that screening of people of Mideastern or mixed ancestry be done with HIM in leukocytes rather than in serum or with the specific substrate for Hex A in serum.

INCREASED SERUM HEXOSAMINIDASE A DURING PREGNANCY: IMPLICATIONS FOR TAY-SACHS CARRIER DETECTION BY SULFATED FLUOROGENIC SUBSTRATES

Carrier detection of Tay-Sachs disease (TSD) in sera is unreliable during pregnancy when based on the heat inactivation method. This is due to an increase in the relatively thermostable serum hexosaminidase (Hex) P, and is overcome by testing leukocytes. Since sulfated fluorogenic substrates (MUβGIcNAc-6-S & MUβGa1NAc-6-S) are highly specific for Hex A and provide direct determination independent of heat-labile/heat-stable ratio, we have examined their value in TSD carrier detection in pregnant women's sera. Determination of Hex activity toward sulfated substrates was carried out in isolated isozyme fractions from sera, leukocytes and fibroblasts of controls, pregnant TSD carriers and non-carriers, and TSD and Sandhoff disease patients. The results indicated that the cleavage en bloc of the N-acetylhexosamine-6-sulfate is catalyzed by an active site on the α-subunit, since only negligible activity was associated with Hex isozymes B, I1, I2 and P, and Hex S (α2) was twice as active as Hex A (αβaβb). The results also indicated that in addition to serum Hex P there is an increase in serum Hex A which starts during the second trimester of pregnancy. We conclude that accurate TSD carrier detection in serum during pregnancy may be possible if gestational age-matched controls are used.

Hexosaminidase A deficiency in adults.

Deficiency of hexosaminidase A (Hex A) in adults was found in 15 individuals from nine unrelated Ashkenazi families; 14 individuals had neurological symptoms, one was clinically intact. Clinical, biochemical and genetic findings are reported and compared to previously reported cases. The clinical picture varied between and within families and included spinocerebellar, various motor neuron and cerebellar syndromes. Psychosis appeared in 30% of cases. Involvement of three generations was recorded in one family. The phenotype appears too variable to serve as a basis for genetic classification. The level of Hex A activity in serum, leukocytes, and fibroblasts of all 14 patients was in the range of Tay-Sachs disease (TSD) homozygotes when measured by the routine heat-inactivation method. More sensitive and direct methods detected some residual activity. Cultured skin fibroblasts of patients synthesize the alpha and beta chain precursors of Hex A of the same molecular weight as that synthesized by normal fibroblasts. However, the amount of the alpha chain precursor is greatly reduced. Mature chains were not detected. The one healthy adult we studied displayed a nonuniform distribution of Hex A; while it lacked activity in the serum it had intermediate activity in fibroblasts and leukocytes.

801 DIAGNOSIS AND CARRIER DETECTION OF TAY-SACHS DISEASE USING SULFATED FLUOROGENIC SUBSTRATES: DIRECT DETERMINATION OF HEXOSAMINIDASE A IN SERUM DURING PREGNANCY AND IN THE PRESENCE OF HEREDITARY HEATLABILE HEXOSAMINIDASE B

4-Methylumbelliferyl derivatives of β-N-acetylglucosamine-6-sulfate and β-N-acetylgalactosamine-6-sulfate were prepared by direct sulfation of the commonly used unsulfated derivatives. Both sulfated substrates were highly specific for hexosaminidase (Hex) A and in fractionated samples more than 97% of these activities was found in the Hex A fraction. The thermolability of Hex B and intermediate forms as found in subjects with hereditary heatlabile Hex B, and the increase in an intermediate Hex form as found in serum during pregnancy (Hex P), had no effect on direct determination of Hex A with the sulfated fluorogenic substrates. Serum and leukocytes from patients with infantile Tay-Sachs disease (TSD), including a TSD patient with heatlabile Hex B, had less than 2% of the respective mean activity of non-carriers. The latter patient was estimated to have 24% Hex A based on the heat inactivation method. Carrier values in serum during pregnancy and in samples with heatlabile Hex B were within the range of other carrier values and clearly separated from the respective non-carrier values. Mean carrier activities were 51-56% of the respective non-carrier means. The values of % Hex A as derived from the ratio between activities toward sulfated and unsulfated substrates were comparable to those obtained by heat inactivation excluding those with heatlabile Hex B. This helps detecting TSD genotypes and discriminates them from Sandhoff disease genotypes.